Studies have shown inflammation is involved in the development of acute aortic dissection (AAD). The hypothesis that white blood cell count (WBCc) on admission may have an impact on the short- and long-term outcomes of type A AAD was tested in a large-scale, prospective observational cohort study.From 2008 to 2010, a total of 570 consecutive patients with type A AAD in Fuwai hospital were enrolled and were followed up. Baseline characteristics and WBCc on admission were collected. The primary outcomes were 30-day and long-term all-cause mortality.9 cells/L . After adjustment for age, sex, C-reactive protein, d-dimer, and surgical intervention, elevated admission WBCc (>11.0 × 109 cells/L) remained an independent predictor of 30-day mortality of AAD . No impact of admission WBCc was observed on the long-term all-cause mortality.During a median of 1.89 years of follow-up, the 30-day and long-term all-cause mortality were 10.7% and 6.5%, respectively. Univariate Cox regression analysis identified admission WBCc as an independent predictor of 30-day mortality when considered as a continuous variable or as a categorical variable using the cutoff of 11.0  × 10In conclusion, elevated admission WBCc may be valuable as a predictor of 30-day mortality, and may be useful in the risk stratification of type A AAD during hospitalization. Some inflammatory biomarkers, such as C-reactive protein (CRP),7d-dimer,9 and fibrinogen10 have been shown to be related with the prognosis in patients with AAD. White blood cell count (WBCc) is a commonly used nonspecific marker of acute inflammatory response and limited studies with relatively small sample size evaluated the impact of WBCc on the in-hospital mortality in patients with AAD but reached inconsistent conclusion.11 Moreover, there is lack of data regarding the association of WBCc with the long-term outcomes in patients with AAD. Therefore, we conducted a prospective observational cohort study with relatively large sample size to evaluate the impact of admission WBCc on the short- and long-term outcomes in patients with type A AAD.Type A acute aortic dissection (AAD) is a devastating cardiovascular condition with high mortality and identification of risk factors for prognosis is of great value for risk stratification in patients with type A AAD. In recent decade, studies have shown that inflammation was not only involved in the pathogenesis of AAD,12 Patients were excluded if they had clear etiology, such as Marfan syndrome, Loeys-Dietz Syndrome, or iatrogenic AAD secondary to cardiac surgery or thoracic endovascular aortic repair, or a history of operation for AAD or chronic dissections. This study was approved by the ethical committees of Fuwai Hospital and all patients provided their written informed consent.A series of consecutive patients who presented to the cardiac emergency center for suspicious AAD were screened from January 2008 to December 2010 in FuWai Hospital . Diagnosis of AAD was confirmed by aorta angiography using multidetector computed tomographic (CT) scanning. The AAD was defined as acute if chest pain or other related symptoms were present <2 weeks before presentation to our hospital. The Stanford type A (DeBakey I and II) dissection was defined as involving the ascending aorta and/or aortic arch according to previously published criteria.d-dimer, and platelet count were also tested simultaneously. We also collected baseline data, including sex, age, duration of pain, and previous medical histories, including hypertension, diabetes mellitus, coronary artery disease, and smoking and drinking status. Other recorded clinical characteristics included baseline vital signs at admission , imaging examinations, medicine treatment, and surgical intervention including emergency and selective surgery. The rationale and strategy of surgical techniques were determined by surgeons in the department of cardiovascular surgery at our hospital.Blood samples were obtained within 5 minutes after admission to hospital, and the total WBCc was measured in an ethylenediaminetetraacetic acid-anticoagulated whole-blood specimen. To address the confounding effect of other inflammatory factors, C-reactive protein (CRP), All patients were followed up during hospitalization and subsequently via outpatient clinical visits or interim telephone calls until the occurrence of death or loss to follow-up. The short-term mortality was defined as death within 30 days after admission to the emergency department. Two cardiologists who were blinded to the study analyses in FuWai Hospital defined the causes of 30-day death as aortic rupture, perioperative death, dissection-related organ ischaemia, or other causes. Long-term mortality was defined as all-cause death after 30 days during follow-up.t test or the Mann–Whitney rank-sum test, as appropriate. Categorical data are presented as the numbers and percentages and were compared using the χ2 or Fisher exact test. Analysis was stratified according to patients with a WBCc >11.0 × 109 cells/L and ≤11 × 109 cells/L as a cutoff vale for normal, according to previous studies.13 Short- and long-term clinical outcomes were determined using the Kaplan-Meier method and compared using the log-rank test. Univariate and multivariate Cox proportional hazards analyses were used to analyze the predictive role of WBCc for short- and long-term mortality with hazard ratios (HRs) and 95% confidence intervals (CIs). Three separate multivariable Cox models were constructed with the WBCc entered as continuous data, using a cutoff value of 11.0 × 109 cells/L, or stratified by tertiles. Other variables selected for testing in the multivariate analysis were those with a P < 0.05 in the univariate models. The proportional-hazards assumption was assessed using the log-minus-log-survival function and found to graphically hold. A P value <0.05 was considered statistically significant. Data were analyzed using SPSS version 19.0 .Continuous variables are presented as the mean ± SD or median and the interquartile range (IQR) according to whether the variable conforms to a normal distribution and were compared using Student A total of 570 patients with CT imaging-confirmed diagnosis of type A AAD were enrolled. All patients had a baseline WBCc available, and the follow-up rates were 99.5% at 30 days and 96.8% at 1 year. The average follow-up period was 1.89 years per subject .9 cells/L or ≤11.0 × 109 cells/L are shown in Table d-dimer, CRP, serum creatinine levels, but had lower platelet counts, platelet-to-lymphocyte ratio, and less frequently received surgical treatment compared with those with normal WBCc .Baseline clinical characteristics of patients stratified by WBCc >11.0 × 10The average hospitalization period was 14 days . The overall 30-day mortality was 10.7% (61/570) and 42 patients died of an aortic rupture into the mediastina or pericardial cavity, 9 patients died during the perioperative period, 6 patients died of heart failure, and 4 other patients died from unknown reasons.P < 0.001). When stratified by WBCc tertiles .As shown in Figure P < 0.001) and as a cutoff value of >11.0 × 109 cells/L . Other risk factors associated with 30-day all-cause mortality included age, neutrophil–to-lymphocyte ratio, admission d-dimer, CRP, Creatinine, while platelet count, and surgical intervention were protectors for 30-day all-cause mortality.The results of univariate Cox regression analysis of predictors of 30-day mortality are shown in Table 9 cells/L increase, the cutoff value of 11.0 × 109 cells/L, and tertiles of WBCc are presented in Table 9 cells/L or stratified by tertiles (the highest tertile) after adjustment for age, sex, and other inflammatory factors. Surgical intervention was an independent protective factor for 30-day death in 3 Cox models.Multivariable-adjusted HRs for 30-day mortality according to per 1.0 × 10A total of 509 patients were discharged from the hospital. During follow-up, the long-term all-cause mortality was 6.5% (33/509). Kaplan–Meier analysis of the long-term all-cause mortality according to the cutoff value of admission WBCc revealed no significant differences between patients with elevated and normal admission WBCc Figure . FurtherThe present study, based on a large sample cohort of patients with type A AAD, highlights the relevance of elevated WBCc on admission with 30-day death. The prediction for increased risk of short-term outcomes by elevated admission WBCc in patients with type A AAD even was not affected by surgical intervention, an important protective treatment strategy for type A AAD. The impact of elevated admission WBCc seems to be limited on short-term outcome of AAD, whereas no association was found between the baseline WBCc and long-term all-cause mortality in patients with type A AAD.14 Inflammation biomarkers are associated with acute-phase reactions, complications, and the prognosis of AAD. Elevated d-dimer level has been reported to be associated with increased in-hospital mortality.15 CRP, a nonspecific, but sensitive inflammatory marker, is associated with increased in-hospital death in type A AAD6 and its peak level is also reported to be associated with the long-term events in patients with type B AAD.7Inflammation is involved in medial degradation of the aortic artery, arterial wall remodeling, and contributed to aortic wall weakness.17 Only 2 studies with small sample size reported discrepant results regarding the association between elevated WBCc and in-hospital mortality.11 In 36 Chinese patients with type A AAD,11 an elevated WBCc (≥12.16 × 1099 cells/L) was associated with an increased risk of in-hospital death . The other study reported no association between admission WBCc and in-hospital mortality in patients with AAD (n = 94).8 However, patients recruited in that study were western with both type A and type B AAD, and the study was a retrospective design with a relatively small sample size. In contrast, our study prospectively enrolled a relatively larger sample size of Chinese patients with only type A AAD. The differences in genetic background, study design, and sample size may partly interpret the inconsistent results. Moreover, our results also confirmed previous findings that elevated admission WBCc was associated with increased short-term death in Chinese patients with type A AAD.11 A novel finding of our study is that the association between short-term mortality and elevated admission WBCc was independent of admission CRP, d-dimer levels, and surgical intervention. As the largest study to date addressing the association of admission WBCc with short- and long-term mortality in patients with type A AAD, the present study suggests the utility of WBCc for the identification of patients with type A AAD at high risk of short-term mortality.An elevated WBCc has been observed in patients with AAD in previous studies.18 atherosclerotic vascular disease,19 and peripheral arterial disease20 independent of CRP and other confounders. Because WBC may activate increased inflammation, endothelial damage, procoagulant effects, microvascular damage, result in release of vasculotoxic factors, and subsequently lead to increased risk of death in cardiovascular disease,21 previous studies suppose that WBCc may play a pathogenic role in vascular diseases. However, the mechanism of AAD might be different from other atherosclerotic vascular disease. In our cohort, only 4.4% patients had concomitant atherosclerosis. An elevated WBCc during acute phase of AAD may just indicate acute inflammatory processes in the dissected aortic wall, the severe extent of aortic damage, and the presence of comorbidities and complications of AAD. Indeed, AAD patients with elevated WBCc had elevated levels of CRP, d-dimer, and creatinine, increased heart rates, and decreased platelet count in our study, which indicated a severe clinical illness condition. Moreover, recent studies have shown that parts of WBC, including neutrophil, lymphocyte, and the ratio of neutrophil and lymphocyte, have some prognostic value in cardiovascular disease.24 However, our study showed that the total WBCc, not the ratio of neutrophil and lymphocyte, was independently associated with the short-term outcome in patients with type A AAD, which may suggest the admission WBCc might be a more useful prognostic parameter compared with the ratio of neutrophil and lymphocyte in patients with AAD. In addition, no relationship was found between admission WBCc and long-term mortality. Taken together, these data support that elevated WBCc may be a sensitive inflammatory marker reflecting severe extent of type A AAD, and then predict increased risk of short-term death of type A AAD.An elevated WBCc on admission might be only a high-risk marker, but not directly be responsible for poor short-term outcomes of patients with type A AAD. High WBCc predicts cardiovascular death in patients with myocardial infarction,25 However, after modernizing and standardizing the operative technique and cerebral protection strategies in the last 2 decades, a significant reduction has been observed in operative mortality from 33.9% to 2.8% and a subsequent improvement in the 5-year survival rates from 55% to 85% for type A AAD.26 Operative mortality and major morbidity could also be significantly reduced with a high-volume multidisciplinary thoracic aortic surgery team.27 The high case volumes of AAD patients in our center (nearly 400 cases per year) help accumulate experience for timely diagnosis of AAD and appropriate medical and surgical treatment, and make the low in-hospital mortality reasonable.29 About 70% of patients in our study received timely repair of the ascending aorta, which ensures postdischarge survival rates of patients with type A AAD.25 In addition, some high-risk patients may have no opportunity to be transferred to our center from local hospitals. Our cohort may consist of relative low-risk patients. Finally, a different genetic background compared with Western populations may partly contribute to the low mortalities of AAD because of the as low as 2.8% and 3.2% operative and in-hospital mortalities reported in other Asian patients with type A AAD.30The 30-day and long-term mortality in our study were relatively lower compared to previous reports of Western populations. The International Registry of Acute Aortic Dissection study reported a relatively high in-hospital mortality (32.5%) and the long-term mortality (16.2%).d-dimer, and platelet count.The present study has several important strengths. First, the reliability of our results was enhanced by our large sample size and the consideration of both short-term and long-term outcomes of AAD. Second, we also considered and adjusted for the impact of surgical treatment and other potential inflammatory biomarkers, including CRP, Several limitations need to be mentioned. First, this is a single-center observational study. Our results may not be generalized to patients in other centers because the patient volume and experiences in medical treatment and surgical intervention of type A AAD might be significantly different between our center and other centers. Second, our study only analyzed the WBCc measured within 5 minutes on admission. The analysis of a series of WBCc measurements at different time points may provide more valuable information for evaluating the prognostic role of WBCc in AAD. In addition, detailed operative data, including operative procedures and perfusion techniques, might also significantly affect early and late survival of type A AAD. Thus, additional research is needed to understand the role of admission WBCc, alone or in combination with other circulating inflammatory biomarkers, in the risk stratification of AAD.In conclusion, elevated WBCc on admission may be valuable for prediction of 30-day mortality in patients with type A AAD. The impact of elevated WBCc on short-term mortality of type A AAD is not affected by surgical intervention. No association was found between admission WBCc and long-term mortalities in patients with AAD. These data suggest the utility of inflammatory markers for the identification of patients with AAD at high-risk of short-term mortality.


Pseudomonas syringae—is a highly thermostable protein that exhibits multifunctional abilities e.g., it elicits hypersensitive response (HR), enhances plant growth, acts as a virulence factor, and forms pores in plant plasma membranes as well as artificial membranes. However, the molecular mechanism of its biological activity and high thermal stability remained poorly understood. HR inducing abilities of non-overlapping short deletion mutants of harpins put further constraints on the ability to establish structure-activity relationships. We characterized HrpZPss from Pseudomonas syringae pv. syringae and its HR inducing C-terminal fragment with 214 amino acids (C-214-HrpZPss) using calorimetric, spectroscopic and microscopic approaches. Both C-214-HrpZPss and HrpZPss were found to form oligomers. We propose that leucine-zipper-like motifs may take part in the formation of oligomeric aggregates, and oligomerization could be related to HR elicitation. CD, DSC and fluorescence studies showed that the thermal unfolding of these proteins is complex and involves multiple steps. The comparable conformational stability at 25°C  of HrpZPss and C-214-HrpZPss further suggest that their structures are flexible, and the flexibility allows them to adopt proper conformation for multifunctional abilities.HrpZ—a harpin from  E. amylovoraPseudomonas syringae, Xanthomonas axonopodis, E. chrysanthemi, E. carotovora, also produce harpins Pseudomonas syringae pv. syringae, secretes HrpZPss, a 34.7 kDa protein, which elicits HR in several plants, including tobacco Pss. Experiments on partial deletion mutation studies revealed that several truncated peptides e.g., peptides containing N-terminal 103 amino acids, C-terminal 214 amino acids of HrpZPss (non-overlapping region) can also elicit a strong HR Harpins are a class of proteins produced by certain Gram-negative plant pathogenic bacteria Pss and other harpins Psp and suggested that the region recognized as an HR elicitor is also indispensable for protein oligomerization, in agreement with our earlier proposal Pectobacterium carotovorum elicits HR in plants but does not form dimers or oligomers Pss, and suggested that some of the leucine zipper-like motifs of HrpZPss are not essential to induce HR in tobacco. The HR induced by the mutants was faster than that of HR induced by intact HrpZPss, possibly due to better exposure of the HR inducing region of the protein, for interaction with the plant receptors.In spite of the lack of significant sequence homology in harpins, they share several common properties such as high content of leucine and glycine, relatively high thermal stability, predominantly helical structure and an ability to form pores in cell membranes Pss and an HR-inducing C-terminal fragment, C-214-HrpZPss using thermodynamic, spectroscopic and microscopic approaches. The results obtained are expected to be useful in understanding the relation between oligomer formation, pore formation and HR elicitation.In light of the above, it remains unclear whether oligomerization is an important characteristic for HR elicitation or not. We, therefore, further characterized HrpZPss and other harpins have been extensively characterized biochemically and by mutational analysis, the factors that contribute to their high thermal stability have not been clearly understood. Differential scanning calorimetry (DSC) is a powerful technique for investigating thermal unfolding of proteins and yields three thermodynamic parameters: the melting temperature (Tm), the change in enthalpy (ΔHc) and the heat capacity change (ΔCp) between the native and denatured states. These parameters can be used to estimate the free energy of stabilization or free energy of unfolding (ΔG), and subsequently, a protein stability curve can be drawn, which describes the temperature-dependent variation of stability Pss and other harpins are not known, our further aims are: 1) to determine the conformational stability of HrpZPss and C-214-HrpZPss, and 2) to understand how the two proteins achieve high unfolding temperature. The results obtained indicate thermal unfolding of these proteins is complex and involves multiple steps and that leucine-zipper-like motifs may take part in their oligomerization.Another important characteristic of harpins is their high thermal stability, which has been exploited to purify harpins from a mixture of other proteins Pss was expressed and purified as described earlier hrpZ nucleotide sequence (642 bp) encoding the polypeptide with C-terminal 214 residues of HrpZPss (C-214-PssHrpZ) was cloned under NdeI and EcoRI sites of pET 28a vector (Novagen). E.coli BL21 (DE3) cells transformed with pET 28a- C-terminal-hrpZ were grown in Luria Bertani (LB) broth containing kanamycin (50 µg/mL) to OD600∼0.5 and induced with 1 mM IPTG. After 3 h of induction, the bacterial cells were pelleted, washed and resuspended in 10 mM sodium phosphate buffer (pH 7.5) and immediately sonicated . The sonicate was centrifuged at 14000 rpm for 20 min to remove cell debris and the supernatant was loaded onto a Ni-NTA matrix (Sigma) and washed with 20 mM imidazole. Bound C-214-HrpZPss was eluted with 200 mM imidazole in phosphate buffer and then subjected to gel filtration on a Superdex 75 column (GE Life sciences), which was pre-equilibrated with 10 mM sodium phosphate buffer (pH 7.5) in order to free it from imidazole. The eluted protein was concentrated using an Amicon filter . Purity of the protein was checked by SDS-PAGE on a 12% gel and by ESI-MS (HrpZy ESI-MS .http://www.jascoint.co.jp), equipped with a Peltier thermostat supplied by the manufacturer. Samples were prepared in 5 mM sodium phosphate buffer (pH ∼7.5) and placed in a 2-mm path length rectangular quartz cell. Spectra were recorded at a scan rate of 20 nm/min with a response time of 0.5 sec and a slit width of 2 nm. Protein concentration was 2.25 µM for measurements in the far UV region (250–190 nm). Each spectrum was the average of 4 accumulations. In order to study the effect of temperature on the secondary structure of the protein, CD spectra were recorded in the far UV region at different temperatures.CD spectra were recorded on a Jasco J-810 spectropolarimeter , equipped with fixed reference and sample cells (0.545 ml each). Sample and reference solutions, prepared in 5 mM sodium phosphate buffer, pH 7.5, were properly degassed with stirring in an evacuated chamber for 5 min at room temperature and then carefully loaded in the calorimeter cells. DSC thermograms were recorded at a scan rate of 60 degrees per hour in the Celsius scale. A background scan collected with buffer in both cells was subtracted from each scan. The temperature dependence of the molar heat capacity of the protein was further analyzed by using the Origin DSC analysis software supplied by the manufacturer.Fluorescence emission spectra were recorded on a Spex Fluoromax-4 fluorescence spectrometer. Slit widths of 2 nm were used on both excitation and emission monochromators. Protein samples (∼0.1 OD at 280 nm) in 5 mM sodium phosphate buffer (pH ∼7.5) were excited at 280 nm and the emission spectra were collected from 290 nm. All the spectra were subjected to buffer-correction before the analysis.AFM measurements were performed as described before Pss was submitted to I-TASSER server (http://zhang.bioinformatics.ku.edu/I-TASSER/) in order to build 3-dimensional structural models. The structures were analysed using PYMOL software.The amino acid sequence of C-214-HrpZPss in order to investigate its thermal unfolding mechanism and thermodynamic stability and compare the results with those corresponding to full length HrpZPss. A heating thermogram of C-214-HrpZPss is given in Pss, taken from our previous study Pss contains a broad asymmetric transition, suggesting the presence of two components  of the thermal transition of C-214-HrpZPss from the difference between post-transition Cp (D) and pre-transition Cp (N) baselines of DSC experiments. The average ΔCp obtained from four DSC scans for C-214-HrpZPss was 0.57±0.035 kcal/mol/°C. Although there could be some uncertainty in estimating ΔCp from baselines of DSC thermograms, this method has been shown to be consistent with other methods of determining ΔCpPss the unfolding transition occurs through an intermediate and the estimated ΔCp for the transition from the native state to intermediate state was 1.48±0.27 kcal/mol/°C. A stable baseline could not be obtained in thermal scans up to 110°C for the transition from the intermediate to fully unfolded structure  and transition temperature (Tm) for individual transitions, obtained from the deconvolution analysis, are listed in Pss, obtained from our previous study Htot) is slightly higher for HrpZPss  as compared to the C-214-HrpZPss , suggesting that deletion of N-terminal part (127 aa) of HrpZPss results in only a nominal decrease in the calorimetric enthalpy.Deconvolution of the thermogram of C-214-HrpZPss was investigated by CD spectroscopy. The far UV CD spectrum of C-214-HrpZPss is shown in Pss (dashed line), taken from our earlier study Pss, whereas full length HrpZPss exhibited two minima centred on 206 nm and 223 nm. These features suggested that similar to full length HrpZPss, C-214-HrpZPss is also a predominantly helical protein. To quantitate the content of different types of secondary structures in C-214-HrpZPss, the CD spectrum shown in Pss, indicating high accuracy of the analysis. The values obtained at 25°C for the different types of secondary structures are: α-helix (36.0%), β-sheet (14.0%), β-turns (19.5%) and unordered structures (30.0%)  . For comelements . The secPss, inferred from the DSC studies, was investigated by CD spectroscopy in order to delineate the underlying structural basis. CD spectra of the protein, recorded at various temperatures, were analysed using the CDSSTR program to monitor temperature-induced changes in the content of secondary structural elements, viz., α-helix and β-sheet. Pss. Up to 40°C, there is no obvious change in the α-helical content, suggesting that no change occurs in the secondary structure below this temperature. Since the Tm of transition 1 was estimated by DSC to be around 40°C, this indicates that during transition 1 there is no detectable change in the secondary structure of the protein. The decrease in helical content above 40°C suggests thermal unfolding of the protein. It appears from this study that during transition 2 major structural reorganization takes place.The non-two-state nature of the thermal unfolding process of C-214-HrpZPss recorded at 25°C under native conditions shows a maximum at 330 nm, indicating that the Trp residue of C-214-HrpZPss is in a hydrophobic environment. HrpZPss, which contains a single tryptophan has an emission maxima at 327.5 nm −, cationic Cs+ and Stern-Volmer analysis to determine quenching constant suggest that tryptophan of C-214-HrpZPss is more exposed to the aqueous environment  of full length HrpZPss (2.79 ns), is also consistent with the tryptophan residue of C-214-HrpZPss being slightly more exposed.The fluorescence spectrum of C-214-HrpZironment . The avePss was also investigated by monitoring temperature-dependent changes in the emission maximum and fluorescence intensity of the protein . To investigate the aggregation state of C-214-HrpZPss in solution and to compare it with native HrpZPss, we have performed gel electrophoresis of the two proteins and a representative gel picture is shown in Pss and C-214-HrpZPss, suggesting that both proteins form polydisperse oligomers, i.e. they exhibit heterogeneity in the oligomerization status. Since C-214-HrpZPss was reported to elicit HR in plants Since native HrpZPss and HrpZPss were also investigated by atomic force microscopy (AFM). Pss and HrpZPss, respectively. Both the images show heterogeneity in size and shape among aggregates of this protein, suggesting the presence of polydisperse aggregates. C-214-HrpZPss forms non-specific oligomers ranging from 20 to 40 nm with occasional occurrence of larger oligomers (up to ∼100 nm) (Pss forms non-specific oligomers of relatively large size (25 to ∼200 nm) with occasional occurrence of even larger aggregates (up to ∼500 nm) . Together, the AFM and gel electrophoresis experiments suggest that both C-214-HrpZPss and HrpZPss form polydisperse oligomeric structures.Morphological features of aggregates formed by C-214-HrpZ∼100 nm) A, where∼500 nm) B. Full Pss in polydisperse aggregated structure (see above), we expected that at least two leucine-zipper-like motifs to be present in C-214-HrpZPss. Examination of the primary structure of C-214-HrpZPss revealed the presence of three leucine-zipper-like motifs: stretch-1, residues 18-25; stretch-2, residues 118-139; stretch-3, residues 160-177. Sequence alignment, shown in Defining the tertiary structure of harpins has been a challenge due to their polydisperse nature in solution Pss could form helical structures located at the surface of the protein, such that their interaction between molecules could result in oligomerization, we carried out computational studies to build a 3-dimensional structural model of C-214-HrpZPss using the I-TASSER server Pss and other harpins Pss, thus obtained shows that the structure of this protein is predominantly made up of helical regions connected by turns and loops  at 25°C was estimated to be 1.04 kcal/mol (2 we assume that there is no significant change in heat capacity (Cp) between transition 1 and transition 2. The first transition was attributed to the dissociation of oligomer with different oligomerization states to dimer Pss exists as polydisperse oligomers and the oligomer nucleates from dimer p approximation is valid. The ΔG3 of HrpZPss was determined using equation 2 and plotted as dash-dotted line  describes the thermodynamic behaviour of the equilibrium of unfolding. The stability curve of a protein can be drawn with confidence using the modified Gibbs-Helmholtz equation. For N↔U equilibrium, where N is the native state and U is the unfolded state, the equation is: kcal/mol . In obtated line . Since wPss and C-214-HrpZPss is predominantly α-helical, although the α-helical content in C-214-HrpZPss (36%) is lower than that in the full length protein (51.5%)  is sufficient to elicit a HR in tobacco. Interestingly, we suggested that amino acid stretch (304-337) of HrpZPph may be responsible for oligomerization and biological activity Pss (C-214-HrpZPss), which elicits HR, should also form oligomers. AFM and gel-electrophoresis experiments were carried out with HrpZPss and C-214-HrpZPss to detect oligomerization. The AFM images of HrpZPss and C-214-HrpZPss suggest the presence of oligomeric structure. The size of individual aggregates is around 20–25 nm for HrpZPss, which is comparable to the reported average hydrodynamic radius (Rh) of 20.5±6.2 nm at 25°C for the native protein h of a monomeric protein with 341 amino acids can be predicted from the following equation CD studies show that the secondary structure of HrpZ (51.5%) . The losh was 2.58 nm and we can approximate that an oligomer with size 20–25 nm may consist of tetramers to decamers depending upon how the monomers are associated. Experimental studies suggest that the aggregated structures of HrpZPss can be as large as decamers, or even up to 16mers Pss correlates well with results reported in the literature. The AFM image of C-214-HrpZPss also suggests oligomer formation. The average size of the oligomer is ∼20 nm, much higher than the predicted Rh from equation 3 for the monomer (2.26 nm). These observations suggest that the aggregated structures are oligomers wherein the number of monomers will be guided by the orientation of monomers in the oligomer. Gel-electrophoresis studies give us a broad smear with multiple underlying bands rather than a single band, further supporting the polydispersity of HrpZPss and C-214-HrpZPss. The question that arises next is, what are the sequence/structural motifs that are responsible for the oligomerization of C-214-HrpZPss?The predicted RPss  can elicit HR, but exhibits defective oligomerization Pss. This mutant corresponding to residues 90-240 of full length HrpZPss is capable of inducing HR Erwinia pyrofolie, HpaG of Xanthomonas axonopodis pv. glycines, HrpZPph of P. syringae pv. phaseolicola indicate that the specific amino acid regions with 13-24 amino acids are responsible for HR elicitation LXXLLXXLX), which contains a high ratio of leucines Pectobacterium carotovorum, that does not form oligomers, can be explained in terms of the role of lipid membrane. It is possible that lipid membrane plays an important role in oligomer formation as there are evidences suggesting that lipid membranes assist oligomerization of proteins The 3-D model of C-214-HrpZPss  consistsPss and C-214-HrpZPss. The thermal unfolding profile of C-214-HrpZPss was complex and composed of two transitions. Since C-214-HrpZPss is a mixture of polydisperse oligomers, we propose that transition 1 is due to dissociation of oligomers. Support for this comes from temperature-dependent CD and fluorescence measurements, which show that major changes in the α-helical content of the protein or the emission maximum of tryptophan residues of C-214-HrpZPss occur only above the midpoint (39.9°C) of transition 1. This suggests that during transition 1 major structural reorganization does not occur and hence transition 1 most likely corresponds to the dissociation of oligomeric structure to dimer. We do not have enough evidence that the intermediate is a dimer or not. Considering that the intermediate of thermal unfolding of HrpZPss is a dimer and the oligomers seem to be structures made up of dimers Pss is also dimer. Transition 2 for C-214-HrpZPss starts at 30°C and ends at 90°C. In this temperature range the dimer unfolds to yield unfolded monomers: the helical content decreases continuously and red-shift in the tryptophan maxima was observed  of the C-214-HrpZPss and HrpZPss at 25°C are ∼10.0 kcal/mol. This suggests that deletion of N-terminal 127 amino acids does not severely affect the stability of the protein, which is also consistent with one of their common features; high thermal stability. Together, apart from oligomerization, we also expect that the structures of HrpZPss and C-214-HrpZPss are flexible, which allows them to adopt an appropriate conformation in order to elicit HR or to form oligomer.The conformational stability .(TIF)Click here for additional data file.S2 FigurePss.MALDI-TOF mass spectrum of C-214-HrpZ(TIF)Click here for additional data file.S3 FigureNative PAGE. Lane 1: HrpZPss. Lane 2: C-214-HrpZPss.(TIF)Click here for additional data file.S4 FigurePss fragment corresponding to residues 90-240 of the full length protein.Primary structure of HrpZMM1, a HrpZ Residues in bold correspond to hydrophobic heptadic amino acids, which could form leucine-zipper-like structures.(TIF)Click here for additional data file.S1 TablePss.Results of fluorescence quenching studies with C-214-HrpZ The extent of quenching achieved by different quenchers and the corresponding quenching constants are given. The final quencher concentration in each case was 0.4 M.(DOCX)Click here for additional data file.S1 MethodAnalysis of fluorescence data.(DOCX)Click here for additional data file.


International travel from low-incidence to high-incidence countries for tuberculosis (TB) is regarded as a risk factor for acquiring TB infection. In this prospective study among long-term travellers we examined the incidence of TB infection using Interferon gamma release assay (IGRA) test and compared these data with results from a visit to the TB department to which all long-term travellers were routinely referred.Immunocompetent adults, travelling for 13–52 weeks to TB-endemic countries, donated blood pre- and post-travel for IGRA. The pre-travel IGRA was only tested in case of a positive IGRA post-travel. Results from their visit(s) to the TB department for TST pre- and post-travel were collected and compared with study results.We found two IGRA conversions in a group of 516 travellers, resulting in an attack rate (AR) of 0.4% (95% CI: 0.5 - 13.9) and an incidence rate (IR) of 0.85 per 1000 person-months (95% CI: 0.1-3.1).We found 5 tuberculin skin test (TST) conversions, resulting in AR of 1.9%  and an IR of 4.26 per 1000 person-months (95% CI: 1.38- 9.94). In our study these converters all had a negative IGRA. One traveller however, who was retested later at the TB department due to a positive TST, then appeared to have seroconverted.The risk of long-term travellers among our study population acquiring TB infection is low. We conclude that post-travel IGRA alone could be used for screening for TB infection among long-term travellers to high-endemic TB countries, but preferably not earlier than 8 weeks after return. One might even argue that IGRA testing should be limited to only those travellers who are going to work in a medical setting. A person with a positive IGRA should be referred to a TB physician for further evaluation.The online version of this article (doi:10.1186/1471-2334-14-515) contains supplementary material, which is available to authorized users. International travel from low-incidence to high-incidence countries for tuberculosis (TB) is regarded as a risk factor for acquiring TB infection. This risk increases with the time spent in a high-incidence country and travellers’ participation in high-risk activities such as working in healthcare settings , 2.A latent TB infection (LTBI) can progress to active, infectious disease. For the majority, the estimated lifetime risk of developing active TB for individuals with an LTBI is 10% within the first years after infection , 4. IdenMycobacterium tuberculosis infection was the tuberculin skin test (TST). TST has several limitations: intra-observer variation in reading the results, limited specificity in BCG-vaccinated individuals, false-positive results due to non-tuberculous mycobacteria, and a boosting phenomenon if TST is repeated. In addition, TST must be read after three days, requiring a second healthcare visit, thus resulting in poor compliance [For a long time the only available test for detecting mpliance .M. tuberculosis, other than those used in BCG vaccination [M. tuberculosis infection. Compared to TST, IGRA has several advantages: only a single patient visit is required, results can be available within 24 hours, and there is no cross reaction with the BCG vaccine or with most non-tuberculous mycobacteria.Interferon gamma release assay (IGRA), commercially available since 2005, is an in vitro T-cell based test which quantifies the immune response of T-cells to in vitro exposure to antigens of cination . A positA gold standard for diagnosis of TB infection is absent, hindering good evaluation of the tests. A review in 2008 investigating the role of IGRAs in screening of travellers, showed too many limitations to come to a recommendation. Thus, the advantage of screening travellers by use of IGRA remains unclear .A previous Dutch study conducted in 1994–1996 demonstrated that the incidence of TB for long-term travellers (3–12 months) based on TST conversions approaches that of the local population when travelling to high-incidence countries [This study was part of a larger prospective mono-centre study among persons attending the Public Health Service (PHS) Amsterdam travel clinic in the Department of Infectious Diseases . All immBefore departure, a standard questionnaire was used to collect data on socio-demographics, BCG-vaccination history, travel history , and travel purpose. The study protocol was approved by the Medical Ethics Committee of the Academic Medical Center Amsterdam. Participants were included after providing written informed consent.Blood samples were taken before and 2–6 weeks after travel.For IGRA the QuantiFERON-TB Gold  was performed following the manufacturer’s instruction and was considered positive if ≥ cut-off point of 0.35 IU/ml. All post-travel samples were tested. Due to financial constraints only in case of a positive post-travel sample the pre-travel sample was tested, which had been stored at −80°C. Positive post-travel samples with negative pre-travel samples were considered conversions.TST was performed according to the Mantoux method with 0.1 ml Purified Protein Derivative (PPD) RT23 . Experienced medical staff of the TB department read the induration between 72 hours and 96 hours. Differences in induration of ≥10 mm between pre- and post-travel TST were considered conversions if pre-travel TST was <10 mm.Although TST is recommended ≥ 8 weeks post-travel, participants in this study were referred to the TB department for a TST at the same time as the post-travel study visit. At the TB department, they were either tested and/or asked to return 8 weeks after travel.Data analysis was performed with SPSS version 19.0.0.1 .Chi-square tests were used to evaluate predictive value for TST or IGRA conversion. Tested characteristics included: age, sex, BCG, reason for travel, healthcare work, travel duration, and primary destination.Incidences were expressed as the rate based on the person-time spent in endemic countries. Person-time denominators for converters were divided in half, since infection was assumed to have occurred halfway during travel. For travellers with a negative TST post-travel and an unknown TST pre-travel, we assumed they were also negative pre-travel. Confidence intervals for both the attack- and incidence rates have been calculated.Between December 2008 and September 2011, 684 persons intending to travel to (sub) tropical countries for 13–52 weeks provided informed consent. Of these, 168 were excluded upon completion of the study  complied with a post-travel TST, however 34 did not return to let their TST result be read.Of the remaining 261 post-travel TST, 8 were considered positive. Of these, pre-travel test results were unknown for 3 (non-compliance), resulting in 5 demonstrated and 3 potential conversions  of 1.9%  for demonstrated TST conversions and an incidence rate (IR) of 4.26 per 1000 person-months (95% CI: 1.38- 9.94).When including potential conversions, we found an AR of 3.1% (8/261) and an IR of 6.76 per 1000 person-months (95% CI: 2.9- 13.3).None of the tested characteristics were predictive for TST conversion.Of the 2 travellers with an IGRA conversion, one had a TST of 8 mm post-travel (unknown pre-travel TST) and the other was negative (2 mm), tested 39 and 37 days respectively after return  had a negative post-travel IGRA.Traveller number 7, who had worked in a hospital in East-Africa, had a negative IGRA 18 days after return. At the TB department 79 days after return the TST was 10 mm. The IGRA at that time was also positive (7.47 IU/ml), and therefore treatment for TB infection was started. This was considered the only confirmed seroconversion; of the remaining 7, none were treated for TB infection.M tuberculosis among long-term immunocompetent travellers to TB-endemic countries; an attack rate (AR) of 0.4% and an incidence rate (IR) of 0.85 per 1000 person-months.Based on the IGRA test, this prospective study found a low incidence of recent infection with Based on TST conversions, we found an AR of 1.9% and an IR of 4.3 per 1000 person-months comparable to an AR of 1.8% and an IR of 3.5 per 1000 person-months in a previous Dutch study, which had an enrolment period from 1994 till 1996 [Results of IGRA and TST were not concordant as has been found in other studies –10. The As already mentioned in the introduction, there are several disadvantages of TST compared to IGRA of which the poor compliance is an important one . In addiThe use of IGRA is already known to decrease the number of people that are recommended preventive treatment for TB infection: in the Netherlands adding the IGRA test to the screening protocol  has led to a decrease of the number of people who are recommended preventive treatment .Evaluation of the TB department’s follow-up of travellers with a negative IGRA, performed within the incubation period, and a positive TST showed that in most cases no IGRA retesting had taken place, either because the TB physician found the traveller at low risk for TB infection or due to non-compliance. However, none of the participants was registered in the national TB register by the end of 2013. The only traveller in our study who was considered to have a true conversion and who was treated for TB was one of the 20 travellers who reported to have been working in a medical setting. Therefore one might argue whether testing for TB should be limited to only those travellers with potential exposure by working in a medical setting in TB endemic countries.We conclude that the risk of acquiring TB infection in immunocompetent long-term travellers is low. A post-travel IGRA alone could be a useful screening tool for TB infection among long-term travellers to high-endemic TB countries, arguably even limited to only those travellers with potential exposure by working in medical settings, enhancing compliance compared to TST. However, the IGRA test should be performed at least 8 weeks after return. A person with a positive IGRA should be referred to a TB physician for further evaluation.


Hoplopleura (family Hoplopleuridae).The suborder Anoplura contains 540 species of blood-sucking lice that parasitize over 840 species of eutherian mammals. Fragmented mitochondrial (mt) genomes have been found in the lice of humans, pigs, horses and rats from four families: Pediculidae, Pthiridae, Haematopinidae and Polyplacidae. These lice, eight species in total, are from the same major clade of the Anoplura. The mt genomes of these lice consist of 9–20 minichromosomes; each minichromosome is 1.5–4 kb in size and has 1–8 genes. To understand mt genome fragmentation in the other major clade of the Anoplura, we sequenced the mt genomes of two species of rodent lice in the genus Ho. akanezumi; each minichromosome is 1.7–2.7 kb long and has 1–6 genes. We identified 34 mt genes on 11 minichromosomes in the rat louse, Ho. kitti; each minichromosome is 1.8–2.8 kb long and has 1–5 genes. Ho. akanezumi also has a chimeric minichromosome with parts of two rRNA genes and a full-length tRNA gene for tyrosine. These two rodent lice share the same pattern for the distribution of all of the protein-coding and rRNA genes but differ in tRNA gene content and gene arrangement in four minichromosomes. Like the four genera of blood-sucking lice that have been investigated in previous studies, the Hoplopleura species have four minichromosomes that are only found in this genus.We identified 28 mt genes on 10 minichromosomes in the mouse louse, Our results indicate that fragmented mt genomes were present in the most recent common ancestor of the two major clades of the blood-sucking lice, which lived ~75 million years ago. Intra-genus variation in the pattern of mt genome fragmentation is common in the blood-sucking lice (suborder Anoplura) and genus-specific minichromosomes are potential synapomorphies. Future studies should expand into more species, genera and families of blood-sucking lice to explore further the phylogenetic utility of the novel features associated with fragmented mt genomes.The online version of this article (doi:10.1186/1471-2164-15-751) contains supplementary material, which is available to authorized users. Hoplopleura and Pterophthirus of the family Hoplopleuridae; genera Haemodipsus, Linognathoides, Neohaematopinus and Sathrax of the family Polyplacidae), and sea lions and seals (family Echinophthiridae). The other major clade includes the lice of humans and gorillas (families Pediculidae and Pthiridae), monkeys (family Pedicinidae), pigs and horses (family Haematopinidae), and rodents . Like Kim[The suborder Anoplura contains 540 species of blood-sucking lice that parasitize over 840 species of eutherian mammals–3. BloodLike Kim, Light eLike Kim in the pPediculus humanus[Pe. capitis, and the human pubic louse, Pthirus pubis[Haematopinus suis, and the wild pig louse, Ha. apri[Ha. asini[Polyplax asiatica and Po. spinulosa[The mitochondrial (mt) genomes of eight species of blood-sucking lice have been sequenced entirely or near entirely in previous studies: 1) the human body louse,  humanus); 2) theus pubis; 3) the Ha. apri; 4) the a. asini; and 5) pinulosa. All of pinulosa, 17. Thepinulosa. Indeed,pinulosa, 15. It pinulosa, 14.Polyplax rat lice (Polyplacidae), whose mt genomes have been sequenced, are from the same major clade of the Anoplura[Hoplopleura (family Hoplopleuridae): Ho. akanezumi and Ho. kitti. Hoplopleuridae is the most species-rich family of the Anoplura with 162 described species, and Hoplopleura is the most species-rich genus in the family Hoplopleuridae with 141 described species found on rodents and pikas[Hoplopleura species are in a major clade different from that of the human lice (families Pediculidae and Pthiridae), the pig lice and the horse louse (family Haematopinidae), and the Polyplax rat lice (Polyplacidae)[Ho. akanezumi infests the Cheverier’s field mouse, Apodemus chevrieri, and five other Apodemus species of mice[. Ho. kitti infests the Bower’s white-toothed rat, Berylmys bowersi, and two other species of rats, Berylmys berdmorei and Leopoldamys edwardsi[Ho. akanezumi and Ho. kitti have fragmented mt genomes. These two rodent lice, however, differ in the pattern of mt genome fragmentation. Like other genera of blood-sucking lice, the Hoplopleura species also have minichromosomes that are only found in this genus.The three human lice (families Pediculidae and Pthiridae), the two pig lice and the horse louse (family Haematopinidae), and the two Anoplura. To undend pikas, 8. Hopl of mice, 18. Hoedwardsi, 18. We Ho. akanezumi, were collected at Cangshan Mountain, Dali city, Yunnan, China (sample No. 249). The Bower’s white-toothed rat lice, Ho. kitti, were collected in Jinping county, Yunnan, China (sample No. 344). The rodents were caught with trap-cages set outdoors . Alive rodents trapped were placed individually in pre-marked cotton bags and transferred to laboratory for species identification and parasitological check. Blood-sucking lice on the body surface of each rodent host were collected and preserved in 95% ethanol at –20°C prior to DNA extraction. Samples of Ho. akanezumi and Ho. kitti and their rodent hosts were deposited in the Institute of Pathogens and Vectors, Dali University. The capture of rodents was approved by health authorities in Yunnan province, China. Animal protocols and procedures were approved by the animal ethics committees at Guizhou University and Dali University .The Chevrier’s field mouse lice, rrnS gene and a 360-bp fragment of mt rrnL gene were initially amplified by polymerase chain reaction (PCR) with primer pairs 12SA–12SB and 16SF–Lx16SR . Two pairs of specific primers for Ho. akanezumi, 12S249F–12S249R and 16S249F–16S249R, were designed from sequences of the rrnS and rrnL fragments. The two specific primers in each pair go outbound and are 1 bp and 89 bp respectively from each other. PCRs with these specific primers amplified two near full-length mt minichromosomes of Ho. akanezumi that contain rrnS and rrnL respectively; these amplicons (1.7 kb and 2.1 kb in size) were sequenced using Sanger method at the TBB. Another pair of primers specific to Ho. akanezumi, 249F–249R, was designed from conserved non-coding sequences that flank the coding regions of the two minichromosomes above. The PCR with 249F–249R primers produced a mixture of amplicons ranging from 0.4 to 2 kb in size, expected from the coding regions of the whole set of mt minichromosomes of Ho. akanezumi . A 452-bp fragment of mt g region–15.FigurHo. kitti, a 532-bp fragment of mt cox1 gene, a 452-bp fragment of mt rrnS gene and a 360-bp fragment of mt rrnL gene were amplified initially by PCR with primer pairs mtd6–mtd11, 12SA–12SB and 16SF–Lx16SR  were sequenced using Sanger method at the TBB. Another pair of primers specific to Ho. kitti, 344F–344R, was designed from conserved non-coding sequences that flank the coding regions of the three minichromosomes above. The PCR with primer pair 344F–344R produced a mixture of amplicons ranging from 0.6 to 2 kb in size, expected from the coding regions of all mt minichromosomes of Ho. kitti  was used in the initial short PCRs with the following cycling conditions: 94°C for 1 min; 40 cycles of 98°C for 10 sec, 45°C for 30 sec, 72°C for 1 min; and a final extension of 72°C for 2 min. LA Taq (TakaRa) was used in the long PCRs with the cycling conditions: 94°C for 1 min; 35 cycles of 98°C for 10 sec, 60–65°C (depending on primers) for 30–40 sec, 68°C for 3 min; and a final extension of 72°C for 6 min. Positive and negative controls were run with each PCR experiment. PCR amplicons were checked by agarose gel (1%) eletrophoresis; the sizes of PCR amplicons were estimated by comparing with molecular markers. PCR products were purified with Wizard SV Gel/PCR clean-up system (Promega).Ho. akanezumi and Ho. kitti were sequenced with Illumina Hiseq 2000 platform at the BGI-HK. Illumina sequence-reads were assembled into contigs with Geneious 6.1.7[Ho. akanezumi and Ho. kitti identified by sequence-read assembly were verified by PCR . All of the 28 mt genes of Ho. akanezumi that we found have the same orientation of transcription relative to the non-coding region , which are only 5% (i.e. 51 bp) and 24% (i.e. 172 bp) of the full-length rrnL and rrnS, respectively. A tRNA gene, trnY2, is upstream prrnL in the chimeric minichromosome and has the same sequence and length as its counterpart in the trnY2-rrnL-trnV minichromosome  is present downstream the 3’-end of the coding region  is present in the NCRs upstream the 5’-end of the coding region, and a GC-rich motif  is present downstream the 3’-end of the coding region in Ho. kitti , rabbits and shrews , and sea lions and seals (family Echinophthiridae). The other major clade includes the lice of humans and gorillas (families Pediculidae and Pthiridae), monkeys (family Pedicinidae), pigs and horses (family Haematopinidae), and rodents . Light et al.[18S, EF-1α, and cox1 gene sequences) of species from eight of the 15 families of the Anoplura and is consistent with an early study by Kim[According to Light et al. and Smitt et al. and Smitt et al. used moly by Kim, which uHoplopleura species and the species from the other major clade to be ancestral to the blood-sucking lice. These characters include atp8-atp6-N, E-cob, cob-S1, I-cox1, D-Y-cox2, R-nad4L-P-cox3, cox3-A, K-nad4, and M-L1-rrnL-V; each of these characters is shared by at least one species from each of the two major clades of the blood-sucking lice (Table The presence of fragmented mt genomes in both major clades of the blood-sucking lice indicates that the most recent common ancestor (MRCA) of these lice already had a fragmented mt genome. Although the data available now are not sufficient yet for us to establish the exact ancestral fragmentation pattern of that MRCA, we can infer that the gene-arrangement characters that are present in the Polyplax rat lice in previous studies. In the human lice, Pe. humanus, Pe. capitis and Pt. pubis, eight pairs of mt genes share stretches of identical sequences, 16–127 bp long, which are much longer than expected by chance, providing unequivocal evidence for recombination between mt genes[Ha. suis and Ha. apri (three pairs of genes[Ha. asini (nine pairs of genes[Po. asiatica (one pair of genes) and Po. spinulosa , the hoof genes), and thof genes). It is e AA-arm, 15. In he D-arm. In additrnL1 and trnL2, longer than expected by chance in Ho. akanezumi and Ho. kitti  and part of rrnL gene  . As discussed above, the findings from the present study and two recent studies[The suborder Anoplura contains 540 species of blood-sucking lice in 50 genera and 15 families. The mon studies, 15 showHoplopleura lice. We did not find nine mt genes that were typical of bilateral animals in Ho. akanezumi: trnF, trnG, trnH, trnL1, trnM, trnQ, nad1, nad3 and nad5. We did not find trnF, trnH and nad5 either in Ho. kitti. These unidentified genes in the Hoplopleura lice are present in the fragmented mt genomes of three human lice, two pig lice, the horse louse, and two Polyplax rat lice[Ho. akanezumi and Ho. kitti may not be conserved in the chromosomes that contain these unidentified genes. We cannot exclude either the possibility that macrochromosomes or the typical mt chromosome of animals may exist in the Hoplopleura rodent lice.The PCR-based strategy we used has its limitations in identifying the complete set of mt chromosomes of the rat lice–15. The Pediculus humanus, in conjunction with PCR-based strategy[A non-PCR-based, shot-gun strategy was used to sequence the mt genome of the human body louse, strategy, 26. Thestrategy whereas strategy, 27. ForHo. akanezumi and Ho. kitti, collected from the Chevrier’s field mouse, Apodemus chevrieri, and the Bower’s white-toothed rat, Berylmys bowersi. Both Ho. akanezumi and Ho. kitti have fragmented mt genomes, with the mt genes that we identified distributed on 10 and 11 minichromosomes, respectively. Ho. akanezumi also has a chimeric minichromosome, which has parts of two rRNA genes and a full-length trnY2 gene. These two Hoplopleura rodent lice share the same pattern for the distribution of the protein-coding and rRNA genes but differ in tRNA gene content and gene arrangement in four minichromosomes. Like other genera of blood-sucking lice that have been investigated in previous studies, the Hoplopleura species have four minichromosomes that are only found in this genus. We conclude that fragmented mt genomes were already present in the MRCA of the two major clades of the blood-sucking lice, which lived ~75 Mya. Intra-genus variation in the pattern of mt genome fragmentation is common in the blood-sucking lice. Such variation provides a novel source of information for inferring genus- and family-level phylogenies of the blood-sucking lice, and for testing the phylogenies inferred from morphology and gene sequences. Future studies should expand into other genera and families of the blood-sucking lice to fully explore the phylogenetic utility of the features of fragmented mt genomes.We sequenced the mt genomes of Hoplopleura rodent lice supporting the results of this article have been deposited in GenBank (accession numbers KJ648922–KJ648943).The nucleotide sequences of the mt genomes of the two PCR primers used to amplify and sequence the mitochondrial genomes of the rodent lice,Hoplopleura akanezumi(Hoa) andHoplopleura kitti(Hok).(PDF 92 KB)Additional file 1:PCR primers used to verify the mitochondrial minichromosomes of the rodent lice,Hoplopleura akanezumi(Hoa)andHoplopleura kitti(Hok).(PDF 71 KB)Additional file 2:Inferred secondary structures of the mitochondrial tRNAs ofHoplopleura akanezumi(Ha) andHoplopleura kitti(Hk).(PDF 136 KB)Additional file 3:Alignment of nucleotide sequences of parts of the non-coding regions upstream (A) and downstream (B) of the coding regions of the 10 mitochondrial minichromosomes and a chimeric mitochondrial minichromosomes ofHoplopleura akanezumi. 249F and 249R are the PCR primers used to amplify the coding regions of all mitochondrial minichromosomes of Hoplopleura akanezumi. (PDF 58 KB)Additional file 4:Alignment of nucleotide sequences of parts of the non-coding regions upstream (A) and downstream (B) of the coding regions of the 11 mitochondrial minichromosomes ofHoplopleura kitti. 344F and 344R are the PCR primers used to amplify the coding regions of all mitochondrial minichromosomes of Hoplopleura kitti. (PDF 255 KB)Additional file 5:


Tokudaia muenninki are expected to be evolutionarily ‘young’, and therefore provide a good model in which to elucidate the early phases of eutherian sex chromosome evolution. Here we describe the genomic evolution of T. muenninki in neo-sex chromosome differentiation.Sex chromosomes of extant eutherian species are too ancient to reveal the process that initiated sex-chromosome differentiation. By contrast, the neo-sex chromosomes generated by sex-autosome fusions of recent origin in T. muenninki male, using 50 BAC clones as probes, revealed no chromosomal rearrangements between the neo-sex chromosomes. Substitution-direction analysis disclosed that sequence evolution toward GC-richness, which positively correlates with recombination activity, occurred in the peritelomeric regions, but not middle regions of the neo-sex chromosomes. In contrast, the sequence evolution toward AT-richness was observed in those pericentromeric regions. Furthermore, we showed genetic differentiation between the pericentromeric regions as well as an accelerated rate of evolution in the neo-Y region through the detection of male-specific substitutions by gene sequencing in multiple males and females, and each neo-sex–derived BAC sequencing.FISH mapping of a Our results suggest that recombination has been suppressed in the pericentromeric region of neo-sex chromosomes without chromosome rearrangement, whereas high levels of recombination activity is limited in the peritelomeric region of almost undifferentiated neo-sex chromosomes. We conclude that PAR might have been formed on the peritelomeric region of sex chromosomes as an independent event from spread of recombination suppression during the early stages of sex chromosome differentiation.The online version of this article (doi:10.1186/s12862-015-0514-y) contains supplementary material, which is available to authorized users. The eutherian Y chromosome was originally homologous to the X chromosome . Since tDrosophila lacks recombination during male meiosis, so they have completely differentiated and largely degenerated Y chromosomes [Sex chromosomes have evolved independently many times from different autosomes in different lineages . In eachomosomes . The sexomosomes –9. In coomosomes . In euthomosomes , 11. Oblomosomes , 13. Preomosomes , 14, 15.Tokudaia muenninki) has neo-sex chromosomes formed by sex-autosome fusions and is therefore an excellent model for studies of the initiation process of sex chromosome differentiation [in situ hybridization (Zoo-FISH) has revealed that a pair of autosomes fused with the sex chromosomes of T. muenninki (neo-X and neo-Y), which are homologous to segments of chromosomes 11 and 16 of mouse [T. muenninki correspond to autosomes in the two other Tokudaia species, Tokudaia tokunoshimensis and Tokudaia osimensis [T. muenninki lineage after it diverged from the last common Tokudaia ancestor. On the basis of the sequence data for mitochondrial cytochrome b (Cytb) and nuclear recombination activating gene 1 (RAG1), divergence times between T. muenninki and the two other species are estimated to be around 1.5–1.7 and 0.6–0.8 million years ago (MYA) ([b) and 0.078 (RAG1) per 12 MY; [T. muenninki  is the only species in the genus Tokudaia that has maintained the Y chromosome, probably through fusion with an autosome [SRY pseudogenes [The Okinawa spiny rat  , suggesautosome . The shoautosome . The shoudogenes . The othudogenes , 21.T. muenninki neo-sex chromosomes as a model. First, we used fluorescence in situ hybridization (FISH) in a T. muenninki male to examine loci order in the neo-X and neo-Y chromosomes, as well as identify their chromosomal rearrangements. To estimate recombination rates along the neo-sex chromosome, the fine approach is to construct a genetic map by genotyping, with a high density of markers and a large number of unrelated individuals [T. muenninki is endangered and has been protected by the Japanese government as a natural treasure since 1972. Therefore, we detected variations in G + C content, which is positively correlated with recombination activity [T. muenninki neo-sex chromosomes, to reveal recombination activity in different chromosomal locations. We also studied male-specific substitutions to identify the genetically diverged region between the neo-X and neo-Y, using partial sequences of 21 genes on three different regions of the neo-sex chromosomes. Furthermore, we determined the neo-X– and neo-Y–linked sequences of 18 genes to elucidate the processes underlying their genetic differentiation. In this study, we show that recombination have been suppressed in the pericentromeric region while recombination have been activated in the peritelomeric region of neo-sex chromosomes, indicating PAR might have evolved during the early stage of sex chromosome differentiation.In the study described here, we applied molecular cytogenetic and genetic approaches to reveal the processes of PAR formation and recombination suppression during early-stage evolution of eutherian sex chromosomes, using ividuals . In singactivity , 13, 23,T. muenninki as probes, to confirm conserved synteny between their neo-sex chromosomes and the corresponding autosomes of mouse and rat. BAC clones used for FISH were screened based on genome information from mouse and rat, using as a reference the neo-sex–linked genes we identified in a previous study [T. muenninki neo-X and neo-Y, respectively, as inferred from the genomes of the two other rodents  clones of us study . Sixteennts Fig. .Fig. 1TwT. muenninki neo-sex chromosomes, respectively, from de novo assembled BAC sequences by performing the exon-intron definition based on the mapped read sequences to the mouse genome  for the combined 17 genes were estimated to reveal the variation in selection pressure on neo-Y genes using “free-ratio” model, which assumes a different dN/dS ratio for each branch. We observed that dN/dS ratio was 2.9 times higher in the neo-Y branch than neo-X branch, and the significant difference between neo-X and neo-Y branches was shown by likelihood ratio test , recombination suppression due to a large autosomal inversion during the past 0.5 million years has led to the formation of the neo-Y diverged from the neo-X [T. muenninki neo-sex chromosomes without distinct chromosome rearrangement are in a very early stage of sex-chromosome differentiation.In agreement with a previous study , our FISnki Fig. . Howeverus study . It is hus study . Recombihe neo-X . CompareT. muenninki neo-sex chromosomes and the corresponding regions of rat chromosome 10 : PAR, autosomes, X chromosome, and Y chromosome; this ranking corresponds to decreasing order of recombination activity [T. muenninki neo-sex chromosomes cannot be explained by these two factors. Changes toward GC-richness and AT-richness were observed respectively in the peritelomeric region, which has high G + C content, and the pericentromeric region, which has low G + C content, relative to the corresponding autosomal regions in the two other species. There was little difference in the CpG → TpG/CpA substitution rate between T. muenninki and the other rodent species. Therefore, it was suggested that the sequence evolution toward GC-richness resulted from the increased gBGC by acquisition of recombination activity in the peritelomeric region of the neo-sex chromosomes. To reveal the recombination activity along the neo-sex chromosome, we need to estimate recombination rates in different regions by substitution-direction analysis as well as other approaches such as construction of a genetic map by genotyping with a high density of markers and a large number of unrelated individuals, single-sperm typing, and genetic maps of three-generation families, if we could correct sufficient number of DNA samples or fresh testis.Over the course of sequence evolution, GC-richness increased in the peritelomeric region and decreased in the pericentromeric region of the neo-sex chromosomes Table . G + C cactivity . This efactivity , 23. gBGactivity , 27. Thiactivity , 25, 28.N/dS ratio in neo-Y than neo-X lineage, suggesting that the neo-Y–linked genes had experienced relaxed purifying selection and/or positive selection  and Y  occurs in 25 % of cells examined and that this involves their heterochromatic arms, while the sister taxon (C. h. pretoriae), which lacks these heterochromatic arms, displays complete synapsis of X2 and Y in 100 % of cells examined [T. muenninki has an X chromosome containing a large pericentromeric heterochromatic region and a Y chromosome with heterochromatin in the proximal region of the short arm as well as the entire long arm [T. muenninki Xp and Yp may be prerequisite for recombination suppression, or can at least accelerate the process in their neo-sex chromosomes.Our results suggested that the recombination was suppressed in the pericentromeric region of neo-sex chromosomes from any cause at least other than distinct chromosomal inversion  and has been protected by the Japanese government as a natural treasure since 1972 [T. muenninki in this study. All the animal experiments in this study were approved by Institutional Animal Care and Use Committee of National University Corporation Hokkaido University and performed in accordance with the Guidelines for the Care and Use of Laboratory Animals, Hokkaido University. This article does not contain any studies with human participants performed by any of the authors.nce 1972 . We perfnce 1972  and usedSacI, and subjected to pulsed-field gel electrophoresis. DNA fragments ranging from 125 to 225 kb were isolated and ligated into vector pKS145. Transformation was carried out by electroporation using Escherichia coli DH10B as a host strain. Ampicillin-resistant transformants were collected and stored in 384-well plates. PCR primer sets were designed against M. musculus gene sequences located on chromosomes 11, 16, and 17 (GRCm38/mm10 provided from UCSC) and the orthologous sequences located on chromosome 10 of R. norvegicus . These primers were used to screen the T. muenninki BAC library using a two-step 3D PCR screening system [BAC libraries were constructed according to previously described procedures . Cultureg system . The priT. muenninki were hybridized to male chromosomes. After hybridization overnight at 37 °C, hybridized probes labeled with biotin 16-dUTP or Digoxigenin (DIG) 11-dUTP by nick translation (Roche) were detected with FITC-avidin or anti-DIG-rhodamine (Roche), respectively, and then stained with 4′, 6-diamidino-2-phenylindole (DAPI). The FISH signals were observed under an Olympus fluorescence microscope BX51 and BX53 (Olympus), and the images were captured with an EXi Aqua digital camera (QImaging) and DP73 (Olympus), respectively.Chromosome preparation and FISH were performed as previously described protocol with slight modifications . All BACBAC sequence was determined by NGS on an Illumina HiSeq 1000 using a 100 bp paired-end protocol or an Illumina MiSeq using a 300 bp paired-end protocol. The DNA library was prepared from seven BAC pools  and E. coli (NC_010473) using the BWA 0.5.9 aligner with the default setting, and then the unmapped BAM file generated by SAMtools 0.1.16 was converted into a FASTQ file using bam2fastq 1.1.0 [de novo assembly was carried out across multiple k-mer values using Velvet 1.0.17  and R. norvegicus  were downloaded from UCSC Genome Bioinformatics (http://genome.ucsc.edu/) and NCBI (http://www.ncbi.nlm.nih.gov/). De novo assembled sequences of T. muenninki BAC clones were annotated by identifying each exon of gene sequences determined by the reference mapping to mouse genome. The accession numbers for 120 neo-sex–linked genes of T. muenninki are listed in Additional file T. muenninki were respectively aligned with the coding and noncoding sequences of homologous genes from both mouse and rat using ClustalX 2.1  as outgroup. The direction of nucleotide substitutions among the three sequences was inferred by the maximum parsimony principle shown in Additional file T. muenninki had a G-allele and the other species shared an A-allele, the substitution direction was inferred to be from A to G in T. muenninki. An unaligned region and repetitive sequences were excluded from this analysis.Custom written C scripts were used to estimate the G + C content and the frequency of each substitution direction in the aligned noncoding and coding sequences among Substitution detection was performed in partial sequences of seven genes from the peritelomeric region, five genes from the middle region, and twelve genes from the pericentromeric region of the neo-sex chromosome   with thever. 4.8 , 42. ThiT. muenninki neo-sex–linked genes determined in this study are available on GenBank (accession numbers AB984629-AB984775). The sequence alignment of each gene among T. muenninki, mouse, rat, and guinea pig used in this study are included in Additional file The sequences of


In this study, we examined the effect of muscle fatigue induced by tetanic electrical stimulation (ES) and submaximal isometric contraction on corticomotor excitability. Experiments were performed in a cross-over design. Motor-evoked potentials (MEPs) were elicited by transcranial magnetic stimulation (TMS). Corticomotor excitability was recorded before and after thumb opposition muscle fatigue tasks, in which 10% of the maximal tension intensity was induced by tetanic ES or voluntary contraction (VC). The participants were 10 healthy individuals who performed each task for 10 min. Surface electrodes placed over the abductor pollicis brevis (APB) muscle recorded MEPs. F- and M-waves were elicited from APB by supramaximal ES of the median nerve. After the tetanic ES- and VC tasks, MEP amplitudes were significantly lower than before the task. However, F- and M-wave amplitudes remained unchanged. These findings suggest that corticospinal excitability is reduced by muscle fatigue as a result of intracortical inhibitory mechanisms. Our results also suggest that corticomotor excitability is reduced by muscle fatigue caused by both VC and tetanic ES. Muscle fatigue is defined as an exercise-induced decrease in maximum voluntary contraction (MVC) force produced by a muscle or muscle group  delivered to a peripheral nerve. The amplitude of motor-evoked potentials (MEPs) elicited by TMS in relaxed muscles increases during intermittent exercise or immediately after fatiguing exercise compared with that of MEPs evoked before exercise; this transient post-exercise MEP facilitation lasts a few seconds . For example, repetitive ES for 2 h  that does not induce muscle fatigue.Declaration of Helsinki and International Code of Medical Ethics of the World Medical Association and was approved by the ethics committee at the Niigata University of Health and Welfare, Niigata, Japan. Informed consent was obtained from all participants in the study.Ten healthy volunteers  participated in this study. None of the participants had a history of neuromuscular or cardiovascular diseases and all gave their written informed consent to participate. The study conformed to the Electromyographic activity was recorded via electromyography (EMG) using surface electrodes placed over the abductor pollicis brevis (APB) muscle of the right hand. The active electrode was positioned over the muscle motor point and the reference electrode was placed over the metacarpophalangeal joint. EMG signals were filtered at 20–500 Hz and sampled at 10 kHz using an analog-to-digital converter .TMS was applied using a Magstim 200 magnetic nerve stimulator  with a figure-of-eight-shaped coil . The coil was placed tangentially to the scalp and held at 45° to the midsagittal line for activating the APB muscle. This site was marked with a skin pencil for future reference. The TMS intensity used was the lowest stimulus intensity that induced MEP with a peak-to-peak amplitude, exceeding 1 mV in the relaxed APB in at least 5 of 10 consecutive trials, and this intensity was kept constant throughout the experiments. MEP amplitude was measured in 12 trials before and after each task and analyzed , with the exception of the maximum and minimum MEP amplitudes.Mmax), with a pulse duration of 0.2 ms. F-wave amplitude and persistence and M-wave amplitude were measured in 50 trials before and after each task and analyzed.Excitability of the spinal anterior horn cells was assessed by F-wave recording from the exercised APB muscle. We simultaneously monitored the functional condition of the neuromuscular structure of APB via supramaximal M-wave measurement. For F- and M-wave measurements, we used an electric stimulator  to deliver square pulse stimuli through a bipolar stimulation probe fixed over the median nerve in the neighborhood of the wrist. The stimulus strength used for F- and M-wave recording was 120% of the stimulus strength required to produce maximum M-wave . Maximum muscle force was measured for 5 s and the stable mean of 3 s of this period was calculated.Experiments were performed with three interventions for the same subject. Each sequence was randomized on the other day to avoid influence of the intervention. Participant was seated in a comfortable position and their right arm was fixed to the armchair to allow the measurement of muscle force during thumb opposition movements Figure . The parDuring the VC task, in which muscle fatigue was caused, isometric contraction to achieve thumb opposition was performed for 10 min at an intensity of 10% of MVC. At that time, muscle force was monitored by a personal computer and the participant was instructed to maintain contraction strength using visual feedback.During the ES1 task, in which muscle fatigue was caused, ES was delivered to the median nerve in the wrist  for 10 min at an intensity of 10% of MVC of thumb opposition.ES2 Task was performed for 8 out of 10 participants to examine whether ES of the degree that muscle fatigue is not caused modulate the excitability of the motor cortex.During the ES2 task, in which muscle fatigue was not induced, ES was delivered for 10 min at an intensity of 90% of the MT.Before each task, we recorded MEP, F-wave and M-wave amplitudes and maximum muscle force. Within 10 min of the end of each task, we recorded MEP, F-wave and M-wave amplitudes and maximum muscle force in the same sequence to allow the comparison of pre- and post-task values Figure . During Post hoc analysis was performed by Bonferroni’s method. Differences were considered significant at p < 0.05 for all analyses.Statistical analysis were performed using PASW statistics software version 18 . Two-way repeated measures analysis of variances [time  × movement type ] were used to compare the changes in MEP amplitude, F-wave amplitude and persistence, M-wave amplitude and maximum muscle force. F = 91.787, p < 0.01), but movement type was not significant  = 0.494, P > 0.05). The time × movement type interaction was significant  = 19.003, p < 0.01). Post hoc analyses showed that the post-task maximum muscle force for the VC and ES1 tasks was significantly lower than the pre-task value  = 8.247, p < 0.01) and movement type  = 17.546, P < 0.01). Further, the time × movement type interaction was significant  = 21.262, p < 0.01). Post hoc analyses showed that after the VC and ES1 tasks, MEP amplitude was significantly lower than the pre-task value  = 0.396, p > 0.05) and movement type  = 0.445, P > 0.05). Further, the time × movement type interaction was not significant  = 0.940, p > 0.05).Figure F = 1.761, p > 0.05, persistence; F = 0.001, p > 0.05) and movement type  = 0.489, P > 0.05, persistence; F = 2.360, P > 0.05). Further, the time × movement type interaction was not significant  = 1.614, P > 0.05, persistence; F = 1.749, P > 0.05).Table Our results indicate that the amplitude of TMS-induced MEPs decreases after tetanic muscle contraction for 10 min, induced by peripheral nerve stimulation, without corresponding changes in supramaximal M- and F-wave amplitude. MEP amplitude also decreases after voluntary fatiguing exercise because of PED. In addition, this MEP depression cannot be induced by ES for 10 min at 90% of the MT without muscle contraction.The amplitude of MEP induced by TMS in the APB muscle was significantly lower after tetanic muscle contraction elicited by ES of the median nerve for 10 min. Because MVC force decreased after tetanic ES compared with that before tetanic ES, we can assume that the APB muscle was fatigued. Several researchers (Ridding et al., It is established that Group III and IV muscle afferents fire during continuous muscle contraction in response to both mechanical and metabolic changes in the muscle; these muscle afferents also alter corticospinal excitability (Gandevia et al., PED occurred after 10% MVC for 10 min without corresponding changes in F- and M-wave amplitude. These results are consistent with previous reports on PED (Brasil-Neto et al., M-wave amplitude remained unchanged after muscle contraction induced by the VC- and ES1 task, despite existing muscle fatigue. Some studies (Sacco et al., α-motoneuron excitability represented by F-wave amplitude and persistence also remained unchanged after fatigue induced by both voluntary and tetanic ES. These results are consistent with previous studies (Brasil-Neto et al., In conclusion, our data show that MEP depression occurs after muscle fatigue induced by both tetanic ES and voluntary muscle contraction and that MEP depression does not occur after ES without muscle contraction. These results lead us to hypothesize that central fatigue at the cortical level after voluntary fatiguing exercise is induced by muscle afferents rather than by a voluntary command from a cortical level higher than M1.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.